Our laboratory is studying the 70-kDa heat shock proteins which act as molecular chaperones, that is, fold and unfold proteins in numerous processes in vivo. One approach we are taking to understanding the mechanism of action of these proteins is to carry out a detailed investigation of the mechanism of action of hsp70 in uncoating bovine brain clathrin-coated vesicles and synthetic clathrin baskets. We previously discovered that a 100 kDa protein cofactor is required for the uncoating process to occur. We have now demonstrated that this cofactor is auxilin, a minor assembly protein present in neuronal cells. Our studies on auxilin suggest that it has limited regions which are homologous to regions of DnaJ, a protein cofactor which has been shown to be involved in presenting substrates to hsp70 both in E. coil and higher organisms. Our data suggests that auxilin may be presenting clathrin baskets to hsp70. We have also demonstrated that, in contrast to earlier findings, neither the clathrin light chains nor the terminal domains of clathrin are required for uncoating. This is a major change in the current view of the mechanism of uncoating by hsp70. In another study on uncoating, we previously found that hsp70 causes an initial burst of uncoating followed by slow steady-state uncoating. We now find that this time course of uncoating only occurs when assembly proteins are present; with pure clathrin baskets the time course of uncoating is linear suggesting that in some way assembly proteins are involved in markedly decreasing the rate of steady-state uncoating. We have also directly studied the effect of DnaJ on hsp70. We previously found that DnaJ causes polymerization of hsp70. We have now found that this polymerization occurs much more rapidly than the activation of the ATPase activity showing that ATP hydrolysis follows polymerization. Our data suggests that polymerization and ATP hydrolysis may occur when DnaJ presents one hsp70 to another rather than presenting a substrate to hsp70. Finally, we previously expressed large amounts of human stress hsp70 in E. coli and found that this recombinant protein uncoats clathrin coated vesicles. We have now modified one of the key residues at the ATP binding site, D10, which is involved in the binding of ATP to the active site through Mg. We have found that this modification reduces the binding constants of both ATP and ADP about two orders of magnitude. Furthermore, with either ATP or ADP bound, this mutant enzyme has almost the same properties as nucleotide-free hsp70 suggesting that, by affecting the ability of nucleotide to bind to hsp70, the D10 mutation, in effect, converts the conformation of the enzyme to that of nucleotide-free enzyme even when ATP or ADP is still bound to it.